It is firmly established that Ras proteins are essential control elements of growth promoting signaling networks. Moreover, the deregulation of Ras signaling has been causally linked to a number of human malignancies. However, our understanding of molecular principles that govern the physiological and pathogenic activities of Ras proteins is still limited. The broad objective of this ongoing research program is to elucidate these principles and to determine how they are functionally linked to the control of cellular homeostasis. During the current funding period, we have described a previously unrecognized mechanism for the preferential partitioning of Ras proteins to the endocytic pathway which involves ubiquitin conjugation. Furthermore, we have shown that this targeting mechanism modulates Ras- dependent signaling. The goal of the studies proposed in the current application is to gain detailed knowledge of the regulation and functional consequences of Ras ubiquitination. We will exploit subcellular, cellular and organismal models to pursue the following aims: 1. To define the molecular machinery that controls Ras ubiquitination. Studies proposed within this aim are directed at the identification and characterization of the enzymes that mediate ubiquitin conjugation to and removal from Ras, as well as the delineation of the spatial regulation of Ras ubiquitination. 2. To establish the functional significance of Ras ubiquitination. Studies proposed within this aim are directed at testing the hypothesis that Ras signaling outcomes are influenced by Ras ubiquitination status both in physiological and pathological settings. Together, these studies will provide new insights into specification mechanisms that govern Ras signaling and may uncover new modalities for therapeutic intervention.